Application
News
No.L442
High Performance Liquid Chromatography
Effect of Column Temperature on Organic Acid Separation
LAAN-A-LC-E218
Shimadzu's organic acid analysis system utilizes ion
exclusion chromatography to first separate the target
components, then introduce a pH buffer solution by the
post-column method to adjust the pH to near neutral,
and detect the organic acids using a conductivity
detector to permit quantitation of the various organic
acids detected.
To increase the peak capacity for separations with large
numbers of components, this system is often equipped
with two columns connected in series. Although the
temperatures of these columns are usually set to the
same value, setting their temperatures to different
values permits adjustment of the separation conditions
to improve the resolution of closely eluting organic
acids. Here we report the results of analysis of a mixture
of organic acids using a dual-column, dual-temperature
method.
n Adjusting Separation by Changing Column Temperature - Part 1
When a sample contains numerous organic acids, it
may be difficult to separate all of the components using
the standard analytical conditions designed for the
organic acid analysis system (Table 1).
In this situation, it may be possible to adjust the
separation of the organic acids from each other by
changing the column temperature and mobile phase
concentration, but in some occasions complete
separation may not be achievable even if the
temperatures of the two columns are changed together
due to differences in the retention behaviors of the
many organic acids.
Fig. 1 shows chromatograms of a standard mixture of
organic acids run at column temperatures of 25 °C and
48 °C. With a column temperature of 25 °C, the peaks
of succinic acid and lactic acid, and of fumaric acid and
acetic acid, respectively, were not completely separated.
However, while the peaks of succinic acid and lactic
acid, and fumaric acid and acetic acid were separated
using a column temperature of 48 °C, the peaks of
phosphoric acid and α-ketoglutaric acid, and fumaric
acid and formic acid were not separated. The
separation status of the various peaks is summarized in
Table 2.
min
mV
10 20 30
0
10
20
1
2
3
4
5
6
7
8
9
10
11
12
min
mV
10 20 30
0
25
1
2
3
4
5
6
7
8
9
10
11
12
■Peaks
1. Phosphoric acid 2.α-Ketoglutaric acid 3. Citric acid
4. Pyruvic acid 5. Malic acid 6. Succinic acid
7. Lactic acid 8. Formic acid 9. Fumaric acid
10. Acetic acid 11. Levulinic acid 12. L-Pyroglutamic acid
25 ℃
48 ℃
Fig. 1 Chromatograms of Organic Acid Mixture at Column Temperatures
of 25 °C and 48 °C
Column : Shim-pack SCR-102H, 2 columns in series (300 mm L. × 8.0 mm I.D., 7 μm)
Mobile Phase : 5 mmol/L p-TSA
pH Buffer Solution : 5 mmol/L p-TSA, 20 mmol/L Bis-Tris, 0.1 mmol/L EDTA-4H
Flowrate of Moble Phase : 0.8 mL/min
Flowrate of pH Buffer Solution : 0.5 mL/min
Column Temp. : 40 °C
Injection Vol. : 10 μL
Detection : CDD-10A
VP
Table 1 Standard Analytical Conditions
